Near-ir indocyanine green doped multimodal silica nanoparticles and methods for making the same

ABSTRACT

The subject invention provides novel fluorescent core-shell nanoparticles comprising an encapsulated fluorescent core comprising an ionically bound fluorescent dye and a metal oxide shell. In one exemplary embodiment of the invention a core containing indocyanine green (ICG) with a silica shell that displays excellent photostability for generation of a near infrared fluorescence signal. The fluorescent core-shell nanoparticle can be further modified to act as an MRI, x-ray, or PAT contrast agent. The ICG nanoparticles can also be used as photodynamic therapeutic agent. Other embodiments of the invention directed to methods of making the novel core-shell nanoparticles and to the use of the core-shell nanoparticles for in vitro or in vivo imaging.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims the benefit of U.S. ProvisionalApplication Ser. No. 61/309,261, filed Mar. 1, 2010, the disclosure ofwhich is hereby incorporated by reference in its entirety, including anyfigures, tables, or drawings.

The subject invention was made with government support under theNational Science Foundation, Contract No. EEC0506560. The government hascertain rights to this invention.

BACKGROUND OF THE INVENTION

Fluorescent dyes are widely used for near-infrared imaging but manyapplications of these dyes are limited by disadvantageous properties inaqueous solution that include concentration-dependent aggregation, pooraqueous stability in vitro and low quantum yield. For example, aparticularly useful and FDA approved dye, indocyanine green (ICG), isknown to strongly bind to nonspecific plasma proteins, leading to rapidelimination from the body, having a half-life of only 3-4 min. Otherlimiting factors displayed by ICG include: rapid circulation kinetics;lack of target specificity; and changes in optical properties due toinfluences such as concentration, solvent, pH, and temperature. Toovercome some of these shortcomings the inclusion of the fluorescentdyes into micellar and nanoparticulate systems have been examined.

Attempts to encapsulate ICG into silica and polymer matrices have beenmet with only partial success. Much of this appears to stem from ICG'scombined amphiphilic character and strong hydrophilicity. It containsboth lipophilic groups and hydrophilic groups that promote itsdistribution at interfaces and its interaction with the surfactants thatare often necessitated in the particles synthesis and largely limits itsincorporation to the interior of nanoparticles. ICG displays a criticalmicelle concentration of about 0.32 mg/mL in H₂O and readily partitionsinto aqueous environments, and, therefore, ICG encapsulation inparticulate matrices suffers from significant leaching.

Nevertheless, encapsulated ICG and other fluorescent dyes remainattractive for bio-imaging techniques that non-invasively measurebiological functions, evaluate cellular and molecular events, and revealthe inner mechanisms of a body. Fluorescent dye comprising nanoparticlesare useful for in vitro fluorescence microscopy and flow cytometry.Additionally, fluorescent dye comprising nanoparticles are potentiallyvaluable for photoacoustic tomography (PAT), an emerging non-invasive invivo imaging modality that uses a non-ionizing optical (pulsed laser)source to generate contrast. A PAT signal is detected as an acousticsignal whose scattering is 2-3 orders of magnitude weaker than opticalscattering in biological tissues, a primary limitation of opticalimaging.

Additionally, diagnosis often necessitates the use of more than oneimaging technique to integrate the strengths of multiple techniques andovercome the limitations of an individual technique to improvediagnostics, preclinical research and therapeutic monitoring. Examplesof PAT complementary techniques include magnetic resonance imaging(MRI), positron emission tomography (PET), X-ray tomography,luminescence (optical imaging), and ultrasound. Typically, analysis bydifferent techniques requires different contrast agents. Furthermore,using multiple bio-imaging techniques requires significantly greatertime and expense, and can impose diagnostic complications. If thefluorescent dye comprising nanoparticles include one or more additionalcontrast agents, multiple bio-imaging techniques could be carried outrapidly or simultaneously. Multi-modal contrast bio-imaging agents arepotentially important tools for developing and benchmarking experimentalimaging technologies by carrying out parallel experiments usingdeveloping and proven techniques.

To these ends, effective and stable fluorescent dye comprisingnanoparticles and methods for their preparation are needed. Such novelnanoparticles could be employed for multiple biological applications,including imaging, even multiple bio-imaging techniques, andtherapeutics.

BRIEF SUMMARY OF THE INVENTION

Embodiments of the invention are directed to fluorescent core-shellnanoparticle wherein a core comprising a water soluble fluorescent dyeis encapsulated in a silica shell. The dye is ion-paired with a cationicpolymer and/or with a multivalent cation as a precipitated non-solublematrix. In an exemplary embodiment of the subject invention, a FDAapproved fluorescent dye, indocyanine green (ICG), is used. In oneembodiment, the cationic polymer is chitosan treated bytripolyphosphate. In another embodiment, the multivalent cation is Ba²⁺and the dye is distributed in precipitated BaSO₄. The novel core-shellnanoparticles can be monodispersed with sizes less than 100 nm.

Embodiments of the invention are directed to methods of making the novelfluorescent core-shell nanoparticle. This is done by using awater-in-oil microemulsion directed synthesis. In one embodiment, thepreparation steps comprise: providing core within the water phase of awater-in-oil microemulsion where the core comprises a polymer havingcationic sites, such as protonated chitosan, and/or an insoluble salt ofa multivalent cation, such as a Ba²⁺ salt with a fluorescent dye havinga plurality of anionic sites, such as ICG, and coating the core with ametal oxide layer, for example a silica layer, by condensation of aprecursor, for example, ammonium carbonate catalyzed condensation ofsilanes.

Advantageously, fluorescent core-shell nanoparticles according toembodiments of the invention display good photostability. The syntheticmethods used for the novel core-shell nanoparticle allow a multisteparchitecture on the nanoparticle, where, for example, the use of bariumsulfate enables CT or X-ray contrast as well as near infraredfluorescence traceability and/or the inclusion of other contrast agentsfor robust multimodal bioimaging.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of chitosan stabilized indocyaninegreen (ICG) dye encapsulated in the silica matrix coated withpolyethylene glycol (PEG) according to an embodiment of the invention.

FIG. 2 is a schematic illustration of the ionic interaction betweenbivalent cation Ba²⁺ and the sulfonate groups of single ICG dianion.

FIG. 3 shows (left) a TEM picture with a scale bar indicating 50 nm forabout 25 nm ICG-BaSO₄ silica nanoparticles according to an embodiment ofthe invention and (right) an energy dispersive X-Ray spectrum thatindicates the constituent elements of the ICG-BaSO₄ nanoparticles.

FIG. 4 shows a visible fluorescence microscopy image (×60) of washedBT474 cells after exposure to ICG core-shell nanoparticles for 24 hoursaccording to an embodiment of the invention where the ICG core-shellnanoparticles appear red (bright) with blue nuclear staining fromHoechst 33258.

FIG. 5 shows photoacoustic images using ICG core-shell nanoparticlesaccording to an embodiment of the invention in (a) tissue like phantomat depth of 1 cm for a 3 μL injection of 3 mg/mL suspension and (b)following an intratumoral injection of 10 μL of a 3 mg/mL suspensioninto a mouse bearing human breast tumor.

FIG. 6 shows photostability of 20 nm (displayed in a TEM image in aninset) ICG-BaSO₄-aminated silica core-shell nanoparticles according toan embodiment of the invention versus free ICG dye on continuousillumination.

FIG. 7 shows photobleaching of ICG core-shell nanoparticles according toan embodiment of the invention and ICG dye on continuous illumination.

FIG. 8 shows fluorescence from (A) ICG core-shell nanoparticlesaccording to an embodiment of the invention obtained aftercentrifugation and re-dispersion in water; (B) supernatant and (C) ICGdye on continuous illumination.

FIG. 9 shows increased photostability of the ICG core-shellnanoparticles according to an embodiment of the invention as compared toICG dye.

FIG. 10 shows the fluorescence emission spectra of ICG core-shellnanoparticles according to an embodiment of the invention and ICG dyewith maxima at 800 nm (710 nm excitation).

FIG. 11 shows the fluorescence emission spectra of the ICG core-shellnanoparticles (dual emission) according to an embodiment of theinvention and ICG dye upon excitation at 475 nm.

FIG. 12 shows visible light fluorescence from multimodal ICG-Gdcore-shell nanoparticles labeled J-774 macrophage cells according to anembodiment of the invention.

FIG. 13 shows multiple fluorescence microscopy images of ICG core shellnanoparticle decorated breast cancer cells using three filter settings:Alexa 488, Alexa 633 and Alexa 750 according to an embodiment of theinvention.

FIG. 14 shows NIR fluorescence (745 nm Excitation; 820 nm Emission) frommultimodal ICG-Gd core-shell nanoparticles labeled cells according to anembodiment of the invention.

FIG. 15 shows MR contrast generated in cells using ICG-Gd core-shellnanoparticles according to an embodiment of the invention, where thelabeled cells can be imaged by T1 (left) and T2 (right) weightedsequences.

FIG. 16 shows (left) real-time imaging using nude mice where tail veinhad been injected with ICG core-shell nanoparticles after 60 minutesaccording to an embodiment of the invention and (right) monitored forover 150 minutes.

DETAILED DISCLOSURE OF THE INVENTION

Embodiments of the invention are directed to fluorescent core-shellnanoparticles containing ionically bound ICG or other fluorescent dyeswhere the dye has at least one anionic site and is included within acore bound within an insoluble difunctional or multifunctional metalsalt or ionically bound to a biocompatible polymer having a plurality ofcationic sites and crosslinked into an insoluble polymer matrix core,and where the core is encapsulated in a metal oxide shell. Otherfluorescent dyes that can be use in place of or in addition to ICGinclude, but are not limited to, Evans blue, bromothymol blue, and roseBengal. For purposes of the invention, the core is a material that isformed in a first step and the shell is a material that is formed in asecond step, and although in many embodiments of the invention the shellmaterial will have limited penetration into the core material, in someembodiments of the invention, the shell material can penetrate deeplyinto or extending throughout the core material, yet the core and shellmaterials remain separate material phases. A simplified schematicrepresentation of the particle design is shown in FIG. 1, where multiplecore particles are dispersed within a metal oxide (silica) matrix withsilica at the surface of the matrix. By including appropriate insolublesalts, the fluorescent nanoparticles can display X-ray, CT, and/or MRIcontrast properties in addition to the fluorescence properties.Insoluble salts include, but are not limited to, barium sulfate, calciumoxalates, calcium fluoride, and ferric orthophosphate. In otherembodiments of the invention, the nanoparticle can be further decoratedto include aptamers, metal speckles, and/or groups to enhancesolubility, affinity, or resistance to absorption or agglomeration ofthe fluorescent nanoparticles for use in a desired environment, forexample in vivo. The ICG or other fluorescent dyes can be fixed withinthe fluorescent nanoparticles in a manner such that the dye can leachinto a tumor or other structure and used as a therapeutic agent.

Some embodiments of the invention are directed to a method of preparingthe novel fluorescent core-shell nanoparticles. The method involvesformation of a core by a water-in-oil microemulsion directed synthesis.The oil can be any water immiscible liquid, for example a hydrocarbonsuch as hexane, cyclohexane, heptane, or iso-octane. The size of thenanoparticle cores formed by this novel microemulsion method can betuned from as little as 5 to 150 nm by controlling the molar ratio ofwater to surfactant and the concentrations of the reagents. The confinedsurfactant stabilized aqueous micelles of the microemulsion allow forthe preparation of nanoparticles that have a very narrow sizedistribution, nearly monodispersed nanoparticles having a maximumpolydispersity index (volume average particle size/number averageparticle size) of 1.2.

In one embodiment of the method, a water-in-oil microemulsion isgenerated where the micelles include the soluble fluorescent dye saltand solubilized chitosan. In other embodiments of the invention, thechitosan can be replaced with other polymers containing primary aminogroups, for example, polyethylenimines (PEI) or polylysine, and can be alinear polymer, a branched polymer, a hyperbranched polymer or adendrimers. The chitosan, or other polymer, can be dissolved in a diluteacetic acid solution and mixed with ICG, generally, but not necessarily,as a disodium salt dissolved in water and mixed with a polyanionicprecipitant, for example the polyacid tripolyphosphate, where theprecipitant forms ammonium cations on the chitosan which formprecipitating ionic cross-links and binds the ICG. A silica shell issubsequently formed about the chitosan containing core by hydrolysis andcondensation of a tetraalkoxysilane, such as tetramethoxysilane ortetraethoxysilane, at the interface of the aqueous micelle containingthe chitosan ICG precipitate. Other silanes that can be combined withthe tetraalkoxysilane include, but are not limited to3-mercaptopropyltrimethoxysilane, 2-methoxy(polyethyleneoxy)propyltrimethoxysilane, andN-(Trimethoxysilyl-propyl)ethyldiaminetriacetic acid trisodium salt. Anaminopropyltrialkoxysilane can be included in the silane mixture topromote encapsulation of ICG and the formation of the silica shell aboutthe chitosan ICG precipitate core and to generate sites on thenanoparticles to which moieties are attached to modify the particles forcell targeting, promotion of particle suspension, or additionallyprovide signals for alternate imaging techniques, such as MRI, X-ray orPAT for multimodal imaging. Metal speckles can also be deposited on thesilica shell.

In another embodiment of the invention, the ICG is combined with aninsoluble multivalent cation salt where, for example, a soluble bariumsalt and ICG are present in the micelle of a water-in-oil microemulsion,and subsequently combined with an aqueous sodium sulfate solutionpresent in the water-in-oil microemulsion, to precipitate a Ba-ICG/BaSO₄salt within the micelle. The barium sulfate, or other multivalent cationsalt, permits formation of BaSO₄-ICG/silica core-shell nanoparticlesthat display CT or X-ray contrast as well as MR fluorescencetraceability. The ionic interaction between a single Ba²⁺ cation and thesulfate groups of ICG is illustrated in FIG. 2. The Ba²⁺ cations and ICGdianions can be associated as the 1 to 1 ion pair shown in FIG. 2, as a2 to 2 adduct, as any polymeric adduct, or any combinations thereofwithin the core-shell nanoparticles according to embodiments of theinvention. The silica shell is formed about this insoluble salt core asabove for the chitosan-ICG/silica core-shell nanoparticle.

The nanoparticle cores within the micelles are coated with a silicashell to form the core-shell nanoparticle having an encapsulated dyecore. Traditional sol-gel silica nanoparticle formation that one mightenvision to coat the core within the micelles of a microemulsion iscatalyzed by NH₄OH. However it has been found that this traditionalmethod can not be applied to the preparation of the novel core-shellnanoparticles according to embodiments of the invention because NH₄OHcauses the degradation of ICG with lose of fluorescence propertiesduring synthesis. The degradation can not be prevented by simply using adiluted NH₄OH solution. It has been discovered that by using NH₄CO₃,rather than NH₄OH, the hydrolysis and condensation of the alkoxysilanesoccurs without dye degradation. For example, approximately 24 hoursafter introduction of the NH₄CO₃ catalyst, silica shells are formed onBaSO₄-ICG or Chitosan-ICG cores to yield the desired novel core-shellfluorescent nanoparticles. FIG. 3 shows the TEM of 20±5 nm BaSO₄-ICGsilica nanoparticles.

The formation of silica nanoparticles by a sol-gel process involves twosteps where hydrolysis of the precursor is followed by condensation tothe nanoparticle. Using ammonium carbonate to catalyze generation ofsilica nanoparticles allows a high level of control over thecondensation step. The use of ammonium carbonate appears to modulate therate of silica particle formation and can affect the extent ofcondensation. The extent of condensation affects the mechanical andchemical stability of the nanoparticles. Hence, the nanoparticle can beformed in a manner that can be broken down (degraded) into smallersilica fragments. The particles can be effectively biodegradable, whichprovides significant advantageous for nanoparticles used for biologicalapplications, such as carriers for diagnostic contrast agents, drugdelivery vehicles, and other applications that employ nanoparticulates.The breakdown of the nanoparticle can be promoted by a biologicalenvironment's pH, temperature, ionic strength t, or other factors. Incontrast, ammonium hydroxide catalyzed silica particle formation largelyresults in non-biodegradable silica particles.

In some embodiments of the invention, aminoalkysilanes, for example3-aminopropyltrialkoxysilanes, can be included with the core material orwith the tetraalkoxysilanes to enhance the ICG encapsulation efficiency.Inclusion of the amine sites in the silica matrix additionally allowsfor inclusion of groups for bioconjugation and targeting capability.Also, the aminoalkyl groups of the silica matrix in the shell's surfacecan be modified with polyethyleneglycol (PEG) or other oligomers orpolymers with a strong affinity for water in some embodiments of theinvention such that opsonization is prevented, allowing increasedcirculation times of the particles upon introduction to an organism. PEGmodification can be carried out by the reaction of anN-hydroxysuccinimide ester (NHS) terminated PEG, or other reactiveterminated PEG polymers, with the aminoalkyl containing silica shell.

To overcome issues associated with carrier particle inhomogeneity andallow for the facile obtainment of tunable monodispersed particle sizesof less than 100 nm, a water-in-oil microemulsion mediated synthesisstrategy is carried out by modification of the process disclosed inSharma et al., Chemistry of Materials, 2008, 20(19), 6087-94; Santra etal., Technology in Cancer Research & Treatment, 2004, 4(6), 593-602;Santra et al., Food and Bioproducts Processing, 2005, 83(C2), 136-40;Santra et al., Journal of Nanoscience and Nanotechnology, 2005, 5(6),899-904; Santra et al., Chemical Communications, 2004, 24, 2810-1, allreferences incorporated herein by reference. For example, encapsulationof the surface active dye ICG in a microemulsion can be carried out asfollows. Chitosan and/or a Ba²⁺ salt are dissolved in the aqueousmicelles of the microemulsion, followed by addition of an ICG comprisingsolution such that the ICG partitions into the micelle. Subsequently aprecipitant, tripolyphosphate for chitosan and/or sodium sulfate forBa²⁺ salt, is added to cause precipitation within the micelle,entrapping ICG. Alternately, precipitation can be carried from ahomogeneous aqueous solution that is subsequently used to form amicroemulsion. Although many microemulsion systems can be used,encapsulation of the dyes occurs effectively in a reverse sodiumbis(2-ethylhexyl)sulfosuccinate (AOT) microemulsion system, and does notoccur as effectively in a common Triton X-100 (TX-100) microemulsionsystem. The precipitate containing micelles are then coated with silicaor another metal oxide layer to encapsulate the dye. Again, a simplifiedschematic representation of a nanoparticle according to an embodiment ofthe invention is shown in FIG. 1.

In embodiments of the invention, the novel core-shell nanoparticlescontaining ICG are fluorescent and are useful for imaging byfluorescence microscopy in vitro and quantitative cellular uptake byflow cytometry. For example, the nanoparticles are found to be non-toxicto cancer cells in vitro and can be taken up by cancer cells such as thebreast cancer cells (BT474), as shown in the fluorescence microscopyimage in FIG. 4.

Photoacoustic tomography (PAT) is an emerging powerful non-ionizing deeptissue imaging technology that offers benefits of both high opticalcontrast and high ultrasound resolution. PAT can image with highcontrast and good spatial resolution. In PAT, NIR pulsed laser light isused to generate ultrasound waves in target structures that are detectedand reconstructed for image generation. This will allow non-invasivequantization of nanoparticle contrast agent concentration inside tumors.It has been demonstrated in preliminary experiments that ICG containingnanoparticles are an excellent in vitro and in vivo photoacousticcontrast agent (FIGS. 5 a and 5 b).

The encapsulation of ICG inside of a solid silica core significantlyenhances the dyes capacity for long term imaging. FIG. 6 demonstratesthat ICG-BaSO₄-aminated silica core-shell nanoparticles not only enablean improved photostability over time in comparison to the free dye, butthat the intensity of fluorescence emission initially increased withtime. Samples containing ICG core-shell nanoparticles and a free ICG dyesolution were adjusted to display equal fluorescence emission levels.The two samples were illuminated at 710 nm for 2 minutes, held in thedark for 1 minute, and imaged and this sequence was repeated 12 times asillustrated in FIG. 7. After the 12 cycles, the exposed ICG core-shellnanoparticles were centrifuged and separated from the aqueous medium.The supernatant and the nanoparticles were imaged after resuspension inwater. As shown in FIG. 8, the ICG dye leaches from the nanoparticlesduring photobleaching suggesting that light triggers the release of thedye from the nanoparticles to provide non-photodegraded ICG uponirradiation. The photoinduced dye release provides high fluorescencefrom the dye newly released from the nanoparticles that retainadditional dye for release on subsequent illumination. This hastherapeutic implications, allowing a controlled/triggered release ofdyes from core-shell nanoparticles. The fluorescence intensity of theICG NPs and dye was studied over 7 days (i.e., 166 hours), as shown inFIG. 9, where irradiation was carried out with only few interruptionsfor fluorescence measurements. As opposed to the dissolved ICG dye, theICG doped NPs shows relatively low initial fluorescence intensity thatincreases through the one week period. Although not to be bound by anyparticular mechanism, the photostability of the ICG encapsulated in thecore-shell nanoparticles is consistent with dye stabilization within thesilica matrix due to inhibition of the diffusion of oxygen that promotesphotodegradation into the nanoparticles, whereas slow leaching of thedye from the NPs results in the increase in fluorescence of a sample asthe concentration of non-degraded dye increases with photo inducedrelease from the core-shell nanoparticles.

FIG. 10 shows similar maxima (805 nm) and spectral shapes, for suspendedICG core-shell nanoparticles and dissolved dye upon excitation at 710nm. The consistency of the maxima suggests that the fluorescenceproperty of the dye is not affected by the encapsulation process. Incontrast, FIG. 11 shows the fluorescence spectra of the dye andcore-shell nanoparticles upon excitation at 475 nm. Whereas the ICG dyesshow two emission maxima, at 564 and 805 nm, the ICG core-shellnanoparticles show three emissions at 515 nm, 590 nm and 805 nm. Thesedifferences are suspected to arise from aggregation of the dyes withinthe core-shell nanoparticles. The visible emission observed at about 600nm is advantageous for the tracking of ICG NPs with commonly availablevisible fluorescent microscopes. The spectral differences allow imagingof the ICG core-shell nanoparticles by visible light emission byfluorescence microscopy, as indicated in FIGS. 3, 12, and 13, as well asimaging by NIR emission as illustrated in FIGS. 13 and 14.

The nanoparticle synthesis can be extended to the formation ofmultimodal nanoparticles that can be simultaneously imaged byfluorescence and, for example, magnetic resonance imaging (MRI), in themanner disclosed in Sharma, et al., “Multimodal Nanoparticles forNon-Invasive Bio-Imaging” International Application No.PCT/US08/074,630; filed Aug. 28, 2008, and incorporated herein byreference. FIG. 15 indicates the ability of the particles to generate MRcontrast using ICG-Gd core-shell nanoparticles.

The ICG core-shell nanoparticles can be use for in vivo imaging as shownin FIG. 16. In this example, 20 nm ICG core-shell nanoparticles wereinjected in the tail vein of the mice. As a control, one mouse (farleft) was given a saline injection of similar volume. All the animalswere imaged using the IVIS imaging system. As seen in FIG. 16, initiallythe nanoparticles are visualized in the tail vein at the site ofinjection and after 150 minutes they are distributed in different organssuch as the liver and spleen, demonstrating that these nanoparticles canbe imaged in vivo and tracked in real time. Real time imaging is usefulfor getting information about the pharmacokinetic distribution of theparticles in vivo. Bio-conjugation with homing ligands can enabletracking accumulation of the particles in tumor region, which can beadvantageous for diagnostics as well as therapeutic applications.Additionally, non-invasive real time tracking of size/surface modifiednanoparticles, or cells labeled with ICG core shell particles, can beuseful to understand many biological processes such as stem celltranslocation.

In another embodiment of the invention, ICG core-shell nanoparticles areused therapeutically, for example, for photodynamic therapy (PDT). PDTemploying ICG core-shell nanoparticles and a laser, for example a diodelaser with a wavelength of 805 nm, can be used to treat: Barrett'sesophagus; early esophageal cancer (adenocarcinoma or squamous cellcarcinoma); obstructing esophageal cancer; persistent or recurrentesophageal cancer; gastric cancer; lung cancer; and/or maculardegeneration.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

1-34. (canceled)
 35. A fluorescent core-shell nanoparticle comprising: acore comprising a water insoluble matrix with an ionically boundfluorescent dye having at least one anionic sites; and a shellcomprising a metal oxide, wherein the nanoparticle is less than 100 nmin diameter.
 36. The nanoparticle of claim 35, wherein the metal oxidecomprises silicon dioxide.
 37. The nanoparticle of claim 35, wherein thewater insoluble matrix comprises an ionically crosslinked biocompatiblepolymer having cationic sites, wherein ion-pairing with the fluorescentdye ionically binds the dye within the polymer.
 38. The nanoparticle ofclaim 35, wherein the water insoluble matrix comprises an insoluble saltof a multivalent cation wherein ion-pairing with the fluorescent dyebinds the dye within the salt.
 39. The nanoparticle of claim 35, whereinthe water soluble fluorescent dye is indocyanine green (ICG).
 40. Thenanoparticle of claim 35, further comprising: a metal deposition on saidshell; at least one moiety that exhibits magnetic properties; at leastone moiety that exhibits paramagnetic properties; at least one moietythat exhibits X-ray opacity; a contrast agent for photoacoustictomography (PAT) imaging; or any combination thereof.
 41. Thenanoparticle of claim 40, wherein the moiety that exhibits magnetic orparamagnetic properties comprises at least one lanthanide or transitionmetal.
 42. The nanoparticle of claim 40, wherein the metal comprisesgold.
 43. The nanoparticle of claim 40, wherein said metal is depositedas discontinuous speckles, wherein the metal and the dielectric corehave an interpenetrated gradient.
 44. The nanoparticle of claim 35,further comprising at least one surface functional group.
 45. Thenanoparticle of claim 44, further comprising at least one biomolecule ortargeting ligand attached to the surface functional group for specifictargeting a tumor cell or other biological tissue.
 46. The nanoparticleof claim 35, wherein the surface functional group comprising a moiety topromote suspension of the nanoparticle in water.
 47. The nanoparticle ofclaim 46, wherein the moiety to promote suspension is derived frompolyethylene glycol (PEG).
 48. A method of making a fluorescentcore-shell nanoparticle according to claim 35, comprising: providing acore within the water phase of a water-in-oil microemulsion comprisingan conically cross-linked biocompatible polymer having cationic sitesand/or an insoluble salt of a multivalent cation and a fluorescent dyehaving at least one anionic sites; adding a metal oxide precursor; andforming a metal oxide shell by condensation of the metal oxideprecursor.
 49. The method of claim 48, wherein the microemulsion is areverse sodium bis(2-ethylhexyl)sulfosuccinate (AOT) microemulsion. 50.The method of claim 48, wherein providing comprises precipitating abiocompatible polymer by a polyacid in the water phase of themicroemulsion containing the dye.
 51. The method of claim 48, whereinproviding comprises mixing a soluble salt of the multivalent cation witha soluble salt containing an anion that combines with the multivalentcation to precipitate the insoluble salt of the multivalent cation inthe water phase of the microemulsion containing the dye.
 52. The methodof claim 48, further comprising attaching at least one surfacefunctional group to the shell.
 53. A method of in vivo and in vitroimaging, comprising: administering to a target a fluorescent core-shellnanoparticle according to claim 35, wherein the core comprises a waterinsoluble matrix with an ionically bound fluorescent dye and the shellcomprises a metal oxide, wherein the nanoparticle is less than 100 nm indiameter; and detecting a signal from the nanoparticle.
 54. The methodof claim 53, wherein imaging comprising fluorescence imaging alone, orin combination with one or more of X-ray, CT, and MRI imaging.
 55. Atherapeutic method, comprising: administering to a target a fluorescentcore-shell nanoparticle according to claim 35, wherein the corecomprises a water insoluble matrix with an ionically bound fluorescentdye and the shell comprises a metal oxide, wherein the nanoparticle isless than 100 nm in diameter; and irradiating the fluorescent core-shellnanoparticle with one or more wavelengths of electromagnetic radiationin the infrared, visible, ultraviolet, or X-ray regions of the spectrum.56. The method of claim 33, wherein the therapy is photodynamic therapy(PDT) wherein the source or irradiation is a laser source.